Thermo scientific hm 325 rotary microtome

Samples of skin tissue were fixed using a mixture of formalin, ethanol, and acetic acid in a volume ratio of 4:1:0.3, incubated into paraffin for 48 h at room temperature, and embedded in paraffin. Then, embedded samples were sectioned with a Thermo Scientific HM 325 Rotary Microtome (Thermo Fisher Scientific, Waltham, MA, USA) and sections of 14 μm thickness were collected. All sections were stained with Masson’s trichrome stain (Sigma-Aldrich, USA), embedded in Canada balsam, and studied with a Carl Zeiss Axio Vert.A1 optical microscope (Carl Zeiss AG, Jena, Germany) with an Axiocam 305 color camera (Carl Zeiss AG, Jena, Germany). Images were processed using Zen 2.3 Blue Edition software (Carl Zeiss AG, Jena, Germany).

48 h after the last MPTP injection, mice were intraperitoneally anesthetized with a mixture of Xylazine (50 mg/kg, Xilagesic 2%, Calier Laboratories) and Ketamine (Imagene 50 mg/kg, Merial) mixture at a ratio of 1:1. The brains were immediately removed and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, 0.1 M, pH 7.4) and stored at 4 °C. After 48 h, samples were washed with PBS and absolute ethanol and were embedded in paraffin blocks. The striatum and mesencephalon were cut in coronal sections (thickness 7 μm) on a microtome (Thermo Scientific HM 325 Rotary Microtome, Thermo Fisher Scientific).
Series of sections were stained with tyrosine hydroxylase (TH) (mouse polyclonal antibody), ionized calcium-binding adapter molecule 1 (Iba-1) (rabbit polyclonal antibody), glial fibrillary acid protein (GFAP) (mouse polyclonal antibody), and (low molecular) calcium-binding protein b (S100b) (rabbit monoclonal antibody).

Forty-eight hours after the last MPTP injection, animals were euthanized. First, degus were anesthetized with isoflurane for blood collection and glucose level measurement. Then, animals were introduced into a CO₂ flux chamber, and brains were removed and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer saline (4% PFA in PBS, pH = 7.4) for 48 h, RT. After, brains were washed in distilled water (10 min, 3 times) and immersed in absolute ethanol until they were embedded in paraffin blocks.
Postmortem analyses were performed in 5 µm coronal brain sections, obtained with a microtome (Thermo Scientific HM 325 Rotary Microtome, Thermo Fisher Scientific, Waltham, MA, USA). The studies were performed in the striatum (at the anterior white commissure level), in the ventral mesencephalon (Substantia Nigra pars compacta, SNpc, and the Ventral Tegmental Area, VTA) and in the dorsal hippocampus. Brain areas were identified according to the mouse brain atlas [63 (link)].